Plant growth promotion and alleviation of salinity stress in Capsicum annuum L. by Bacillus isolated from saline soil in Xinjiang

Ecotoxicology and Environmental Safety 164 (2018) 520–529

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Plant growth promotion and alleviation of salinity stress in Capsicum annuum L. by Bacillus isolated from saline soil in Xinjiang

T

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Wenfei Wang, Zhansheng Wu , Yanhui He, Yuanyuan Huang, Xuan Li, Bang-Ce Ye School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832003, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: PGPR PGP properties Capsicum annuum L. Salt stress mitigation

To maintain the growth and development of pepper in saline condition, candidates of plant growth promoting rhizobacteria (PGPR) were isolated, and detected to plant growth promoting (PGP) potential under salt stress was investigated. Thirteen bacterial strains with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, WU-1–13, were isolated from saline soil in Xinjiang, China. The isolates were shown to belong to the genera Bacillus by partial sequencing analysis of their respective 16 S rRNA genes. Seven isolates had the ability to solubilize phosphate. Moreover, the amount of solubilized phosphate was significantly high (P < 0.05), which ranged from 157.33 μg/mL to 922.41 μg/mL. All tested bacterial strains were shown to produce a large amount of ACC deaminase and NH3. Furthermore, nine strains were detected for siderophore production. On the aspect of extracellular enzyme, all bacterial isolates produced lipase, amylase and cellulose, whereas only a minority produced chitinase (15.4%) and 10 isolates produced β-glucanase or protease. In growth room experiments, the results showed that the strain WU-5 exhibited better growth promotion of pepper seedlings in terms of fresh weight (75.60%), dry weight (86.68%), shoot length (12.12%) and root length (146.52%) over the control under saline stress followed by WU-13. Furthermore, seedlings accumulated high amounts of proline induced by the different PGPR inoculation treatments to alleviate the negative effects of salt stress. Further growth-promoting assays under different salt stress were set up to confirm that the fresh and dry weight, shoot and root length of pepper plants inoculated by three strains all were significantly higher than non-inoculated control under different saline stress. In summary, the results demonstrated that WU-9, which induced high levels of proline production and antioxidant enzyme activities, and three strains (WU-5, WU-9 and WU-13) can be of great value in maintaining the growth and development of seedlings on saline lands.

1. Introduction

most to the increasing amount of chemical pollutants via the excessive use of synthetic chemical fertilizers and pesticides, which leads to further environmental damage with potential risks to human health (Ghyselinck et al., 2013). The increasing demand for crop production has become a big challenge at present with a significant reduction of synthetic chemical fertilizers and the adverse effects of saline stress (Vejan et al., 2016). Plant growth promoting rhizobacteria (PGPR) are an important group of microbial communities that exerts beneficial impacts on plant growth and development (Li et al., 2016). Kloepper and partners coined the term PGPR in 1978, and ever since, PGPR have been increasingly seen as a way of complementing conventional inputs and alleviating environmental stress in agricultural systems (Pinter et al., 2017; Kloepper and Schroth, 1978). PGPR widely colonize the root surface and enhance the seed emergence, plant biomass and crop yield (Kloepper and Schroth, 1978). Moreover, the results of plant growth

Xinjiang province, located in northwest China, is a semi-arid region with a temperate continental climate. Given the long-term drip irrigation under mulch, insufficient leaching of salt in soil leads to soil salinization in Xinjiang province. The continuous accumulation of salt in the tillage layer of soils has caused a series of serious adverse effects to crop production. Plant growth is affected by a number of factors, such as nutritional imbalance, hormonal, ion toxicity, and physiological disorders under salt stress conditions (Nadeem et al., 2014; Schoebitz et al., 2013). Salinity also leads to impaired Na+/K+ ratio and osmotic stress, bringing about the inhibition of many biochemical and physiological processes of plant growth and development (Parida and Das, 2005). Nowadays, soil salinity has become one of the naturally occurring problems in various parts of the world (Nadeem et al., 2014). Nowadays, agriculture is one of the human activities that contributes

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Corresponding author. E-mail address: [email protected] (Z. Wu).

https://doi.org/10.1016/j.ecoenv.2018.08.070 Received 7 February 2018; Received in revised form 10 August 2018; Accepted 19 August 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.

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2. Materials and methods

promotion depend mainly on a wide variety of mechanisms, such as organic matter mineralization, biological nitrogen fixation, and biological control against soil-borne pathogens (Kloepper and Schroth, 1978; Pii et al., 2015). Millions of tons of synthetic chemical fertilizers are added to soils every year (Simpson et al., 2011). While a large fraction of them are not utilized by plants, as up to 0.4–90% of the added phosphorus (P) are converted into compounds with low solubility and 50% of the added nitrogen (N) can run off from crop fields (Costa et al., 2012; Simpson et al., 2011). Fortunately, PGPR can have an impact on the availability of the nutrients in soil through the exudation of organic acids (Pii et al., 2015). In addition, several mechanisms of PGPR are presented to promote growth in plants, which include nitrogen fixation, phosphorous solubilization, production of NH3, indole-3-acetic acid (IAA), and siderophores, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, phytohormones production, triggering plant systemic responses and antioxidative enzymes production (Delaplace et al., 2015; Vejan et al., 2016). For instance, plants under heavy environmental stress increase their antioxidant enzyme activities or proline content after inoculation with PGPR to increase plant biomass production and ameliorate the toxic effect (Bharti et al., 2014; Nadeem et al., 2014). Given their beneficial effects on growth and development of host plants, PGPR have an application potential in sustainable, environmental friendly, and organic agriculture (Delaplace et al., 2015; Vejan et al., 2016). Therefore, diverse PGPRs are being used worldwide as bio-inoculants to promote plant growth and development under normal and salinity stress conditions (Bharti et al., 2014; Kamran et al., 2016). Bacteria of the genus Bacillus have been widely reported as good candidates for bacterization because of their ability to eliminate or alleviate the harmful effects of saline stress, regulate plant physiological characteristics and promote plant growth (Pinter et al., 2017; Ghyselinck et al., 2013). For instance, the growth of plumule and radicle of rice seedlings was significantly stimulated by two salt tolerant strains from Bacillus cereus under saline stress ranged from 0 ds/m to 15 ds/m (Kumar et al., 2017). Bacterial volatiles and secretion of B. cereus AR156 promoted the shoot growth of Arabidopsis thaliana wild type and Atabcg30 (Zhou et al., 2015). The highest promotion rate for biomass yield of elephant grass was 116.01% and 81.72% for shoot fresh weight and dry weight, respectively, after inoculation with four mixtures that including Bacillus sp. pp04 (Li et al., 2016). Certain species of the genus Bacillus can promote plant growth and health under normal or adverse conditions, and they are considered to be PGPR. Although many PGPR are described as successful in plant growth promotion, gaps still exist in terms of screening indigenous PGPR in Xinjiang and the importance of different mechanisms in saline stress alleviation, which requires attention from the scientific community (Bharti et al., 2014; Ghyselinck et al., 2013; Li et al., 2016). Therefore, this study attempts to shed more light on isolating PGPR from saline soil in Xinjiang province and testing their growth promoting ability on pepper under salt stress. At the same time, we focus on evaluating the potential use of rhizobacteria as biofertilizers and the relationship between the change of plant biomass and regulation of plant systemic responses in seedlings under salinity stress. Keeping this in view the objective of this study was aimed to (i) identification of 13 bacterial strains by 16 S rRNA gene sequencing; (ii) in vitro characterization of isolated bacterial strains for plant growth promoting properties and extracellular enzyme activity; (iii) evaluation of bacterial strains in increasing biomass of seedlings in pots under saline stress; (iv) characterization of the effects of PGPR candidates on antioxidant enzyme activities and contents of chlorophyll, protein, and proline in bacterized in vitro-grown pepper supplemented with NaCl; and (v) speculation of the main mechanisms of PGPR candidates in promoting plant growth and alleviating salt stress.

2.1. Isolation of ACC deaminase-producing rhizosphere bacteria The bacterial strains were isolated from the rhizosphere of healthy pepper growing in salinized soil of Shihezi, Xinjiang, China (44°27′N; 85°94′E). The ACC deaminase-producing bacteria were isolated based on the method described by Penrose and Glick (2003) and Wu et al. (2012). Single individual colonies of bacteria were maintained as glycerol stock (20%) at − 80 °C for further use. 2.2. Identification of selected rhizobacteria Genomic DNAs of 13 bacterial strains were isolated by using Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, China). For identification, 16 S rRNA gene was amplified in a polymerase chain reaction with universal primers (27 F: 5’-AGAGTTTGAT CCTGGCTCA-3’ and 534 R: 5’-ATTACCGCGGCTGCTGG-3’ synthesized at Sangon Inc., China) using genomic DNA as template (Heyrman and Swings, 2001). Amplification of 16 S rRNA gene sequencing was performed using Ready-to-Use PCR Kit (Sangon Inc., China). The PCR amplicon was purified with SanPrep Column DNA Gel Extraction Kit and sequenced (Sangon Inc., China). The nucleotide sequences were compared against GenBank database using the BLAST tool in the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST). Selected isolates were identified according to 16 S rRNA gene sequences (Pinter et al., 2017; Ghyselinck et al., 2013). Isolates were incubated in nutrient agar for 24 h at 28 °C and observed for morphological characteristics, such as Gram staining, spore, and motility (Harley and Prescott, 2002). Biochemical characterization was followed by testing enzymatic activities, such as catalase (CAT), Methyl Red (M·R), Voges-Proskauer (V-P) tests (Shivsharan et al., 2013). Similarly, isolates were subjected to physiological biochemical tests such as citric acid and malonate. Carbohydrate utilization efficacy of isolates for various sugars, such as lactose, sucrose, maltose, mannitol and xylose was also determined (Harley and Prescott, 2002). 2.3. Direct plant growth promoting (PGP) properties in selected bacterial isolates 2.3.1. IAA production IAA production was measured according to Libbert and Risch (1969). Bacteria were cultivated in fresh Landy medium supplemented with 3 mM L-tryptophan at 28 °C for 48 h. Then cells were separated from culture medium by centrifugation (12000×g, 5 min). One milliliter of the supernatant was mixed with 1 mL of Salkowski's reagent (0.5 M FeCl3 35% HClO4, 2:100), and the mixture was left in the dark for 30 min at room temperature. The absorbance was detected at 535 nm. 2.3.2. Nitrogen fixation The nitrogen fixing capacity was determined by grown on agar plates with N-free semisolid medium NFb (Döbereiner, 1988). Strains were incubated at 28 °C for 7 days. The appearance of a blue halo was observed as qualitative evidence of atmospheric nitrogen fixation. Isolates that exhibited blue zones on the agar plates were selected as nitrogen fixation strains for further study (Li et al., 2016). Performance of nitrogen fixations in liquid nitrogen free Ashby medium was measured according to Wen et al. (2017). 2.3.3. Phosphate solubilization The selected bacteria were grown in the National Botanical Research Institute's phosphate medium (NBRIP) for assessing their ability to solubilize phosphate (Pérez et al., 2007). The clear haloes around the colonies were measured every 24 h for 10 days. Further investigation on phosphate availability was measured in liquid culture 521

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Xinjiang Changji City Xinke Seed Co., Ltd, were employed in this study. A pot experiment was set up in growth chamber to assess the growth promotion effects of the selected isolates inoculated on pepper. Pots were sterilized with 20% sodium hypochlorite solution and then thoroughly washed with sterile distilled water. Round pots containing autoclaved perlite in vermiculite (2:3) were used to grow seedlings. Suspensions of bacterial strains were prepared by shaking cells for 24 h at 28 °C in liquid nutrient medium, and then cells were collected by centrifugation for 10 min at 12000×g and suspended in sterile distilled water. Subsequently, bacterial suspension was used for seed inoculation whereas the non-inoculated control received sterile distilled water treatment. Pepper seeds were soaked in 70% ethanol for 5 min followed by rinsing with sterile distilled water (cycle operation two times) for surface sterilization. The sterilized seeds were thoroughly soaked in 5 mL selected bacterial suspension (2 × 108 CFU/mL) for 5 min. Ten seeds were sown in each pot with eight repetitions, amended with 100 mL of Hoagland's nutrient solution (Hoagland and Arnon, 1950). The seedlings were grown under room condition with temperatures ranging from 26 to 30 °C, 14/10 h light (350 μmol m−2 s−1)/dark period, and a relative humidity of 50–60%. Five milliliters of selected bacterial suspension (2 × 108 CFU/mL) was injected into substrate around the two-week-old seedlings whereas the same amount of sterile distilled water was used as control. Subsequently, four pots of each isolate treated were daily irrigated with sterile water for the first two weeks after germination of seeds. Thereafter, they were supplemented with salt solution (60 mM NaCl) until plants were harvested. The other four pots at zero salinity level were watered with sterile water not supplemented with NaCl. The effect of each isolate was confirmed by measuring seedling biomass (expressed as plant weight and length), chlorophyll, soluble protein, antioxidant enzyme activities and protein content three weeks after the salinity challenge. The detections mentioned above should be repeated four pots for each treatment and sampled three times for each pot.

according to Pereira and Castro (2014). 2.3.4. ACC deaminase activity ACC deaminase activity was determined quantitatively through a colorimetric microplate assay according to the method described by Penrose and Glick (2003). Total protein content was determined based on the method of Bradford (1976), with a standard curve of bovine serum albumin ranging between 0 and 4 mg/mL, by measuring the absorbance at 595 nm. 2.3.5. NH3 production The selected bacteria were grown in peptone water at 28 °C, 200 rpm for 3 days. NH3 production analysis was determined with a Kjeldahl apparatus (Foss Tecator Kjeltec 2300 Analyzer Unit 2300). 2.4. In vitro characterization of biocontrol factors of bacterial isolates 2.4.1. HCN production HCN production was evaluated on nutrient agar amended with glycine (4.4 g/L) medium according to Bakker and Schippers (1987). A piece of filter paper soaked in a 2% sodium carbonate in 0.5% picric acid (yellow) solution was placed on top of each plate. Thereafter, the plates were inverted and sealed with parafilm and incubated at 28 °C for 4 days. Discolouration of the filter paper to orange or red color indicated microbial production of HCN. 2.4.2. Siderophores production The production of siderophores was evaluated on CAS-agar medium according to Schwyn and Neilands (1987). Bacterial plates were incubated at 28 °C for 7 days. The appearance of an orange halo around bacterial colonies (indicating iron chelation) was evaluated to check for siderophore production. 2.4.3. Screening of salt stress alleviating bacteria for extracellular enzyme production Chitinase activity was determined in modified nutrient agar supplemented with colloidal chitin. Bacterial isolates were incubated for 7 days at 28 °C. The ability of chitinase production was shown by a clear halo around bacterial colonies (Kammoun et al., 2008; Yuttavanichakul et al., 2012). Glucanase activity was assayed according to Renwick et al. (1991). After three days of incubation at 28 °C, the plates were stained with Congo Red (0.6 g/L) for 90 min at room temperature. A clear yellow/ orange halo around the isolates was considered as positive for glucanase activity. Protease activity of the bacterial isolates was determined in nutrient agar medium amended with 1% of skim milk (Pereira and Castro, 2014). Isolates were incubated at 28 °C for 3 days. The clear halo around the cells indicated positive proteolytic activity. Cellulase activity was determined according to Kasana et al. (2008). After three days incubation at 28 °C, plates were flooded with Gram's iodine (2.0 g KI and 1.0 g iodine in 300 mL distilled water) for 5 min. A clear halo was considered as positive for cellulase activity. Amylase activity was determined according to Kammoun et al. (2008). After incubation for 3 days at 28 °C, the plates were flooded with Gram's iodine for one minute. A clear zone around the cells indicated positive for amylase activity. Lipases activity was assayed in LB modified agar medium amended with 2.0 g/L of CaCl2 and 10 g/L of Tween-20, Tween-40 and Tween-80 (Pereira and Castro, 2014). Colony growth and halo formation were considered as positive for lipase activity.

2.5.2. Chlorophyll and soluble protein contents of seedlings Chlorophyll content of seedlings was measured with a chlorophyll meter (OPTI-sciences CCM-200 PLUS Leaf Chlorophyll Meter). Solution protein concentration was determined based on the coomassie brilliant blue G-250 method of Bradford (1976) by measuring the photometric absorbance at 595 nm. The soluble protein concentration was calculated using the standard curve of bovine serum albumin (BSA) and expressed as mg protein/g fresh weight. 2.5.3. Antioxidant enzyme activities and proline content of seedlings Peroxidase (POD; EC. 1.11.1.7) activity was measured with 50 mM of phosphate buffer pH 6.0 containing 2.4 mM H2O2 and 20 mM guaiacol as a reaction medium (Zhang and Kirkham, 1994). The kinetic evolution was observed at 470 nm for 2 min. Enzymatic activity was expressed as enzymatic unit (EU) corresponding to 1 μM product /min, extinction coefficient ɛ = 26.6 mM−1 cm−1 (Basiglini et al., 2018). Polyphenol oxidase (PPO; EC. 1.10.3.1) activity was measured by monitoring the decline at 398 nm as catechol catabolization according to Verma et al. (2016). The activity is expressed as enzymatic unit (EU) corresponding to 1 μM product /min, extinction coefficient ɛ = 1.150 mM−1 cm−1 (Basiglini et al., 2018). Proline contents in seedlings were determined according to Bates et al. (1973). Free proline content was extracted from 0.5 g plant material in sulfosalicylic acid 3% w/v for 10 min at 100 °C, then 2 mL of glacial acetic acid and 2 mL of ninhydrin solution 2.5% w/v were added into 2 mL of supernatant and kept in boiling water for 30 min. After cooling, 4 mL of toluene was added to the mixture. Proline contents were estimated by the photometric absorbance of the ninhydrin reaction at 520 nm according to Bates et al. (1973). The proline concentration was calculated from the standard curve of L-proline and expressed as μg proline/g fresh weight.

2.5. Bacterization of isolates in vitro-grown seedlings 2.5.1. Growth-promoting assays Capsicum annuum L. seeds (variety Honglong 23), provided by 522

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and NH3 and ACC deaminase production (Fig. 1). The results showed that all bacterial isolates possessed at least two important directly PGP properties by a series of evaluations. Only the WU-4 strain was positive for IAA production at 4.14 ± 0.05 μg L−1. Two isolates, WU-6 and WU-11 grown in NFb agar plates with a blue halo and exhibited different levels of nitrogen fixation activities ranging from 0.79 to 0.86 mg/L in the Ashby nitrogen-free culture medium (without agar). Seven of the isolates showed different abilities to solubilize phosphate based on the formation of visible dissolution halos on NBRIP agar medium (Table 2). The seven isolates that exhibited clear zones on the agar plates were selected as phosphorus solubilizing strains for further study in liquid culture (Fig. 1). The phosphorus solubilizing bacteria demonstrated a wide range (157.33–922.41 μg/mL of culture filtrate) of soluble-P concentration at 96 h. The bacterial isolates like WU-9, WU-5, WU-2 and WU-6 showed significantly higher (P < 0.05) amount of inorganic phosphorus solubilization, 444.83–922.41 μg/mL of culture filtrate, compared with other bacterial strains. All isolates could successfully grow in the sterile DF salt minimal medium containing 3.0 mM ACC (instead of (NH4)2SO4) as the source of nitrogen, however, they exhibited different levels of ACC deaminase activities (Fig. 1A). The highest ACC deaminase activity was recorded in WU-9 (7.32 μmol α-ketobutyrate (KB)/mg/h) followed by WU-7 (43.13 μmol α-KB/mg/h) and WU-11 (37.92 μmol α-KB /mg/h). Surprisingly, all selected strains also exhibited relatively high levels of NH3 production, with WU-12 having the highest NH3 production at 723.56 mg/ L followed by WU-13 at 702.74 mg/L and WU-2 at 691.55 mg/L (Fig. 1C).

2.5.4. Further growth-promoting assays under different salt stress In order to further assays the growth-promoting under different salt stress, three strains (WU-5, WU-9 and WU-13) were further selected to test the ability to promote pepper growth in pot experiments at different NaCl concentrations (0, 100, 200 and 300 mM) under greenhouse condition. The different treatments should be repeated four pots for each treatment. At seven days of germination, different concentrations of NaCl solutions were applied to the corresponding potted plants. In each treatment, fifteen seedlings were uprooted 3 weeks after germination. The morphological characteristics such as dry and fresh weight, shoot and root length of each plant were recorded. 2.6. Statistical analysis The collected data were analyzed by one-way ANOVA using SPSS version 17.0, and the means were compared using LSD Fisher's least significant difference with a significance level of 0.05 to evaluate the plant growth promotion effects of selected isolates on seedling growth parameters. In plant-bacteria interaction assays, a factorial arrangement of treatments was used with 14 bacteria levels (Control without bacteria and thirteen isolates), 2 salt stress levels (pots irrigated with/ out 60 mM NaCl), 4 pots (repeat four pots per treatment) and 3 replicates. 3. Results 3.1. Identification of selected rhizobacteria Thirteen bacterial strains with ACC deaminase activity were isolated from the rhizosphere of healthy plants growing in saline soil of Xinjiang province. Subsequently, they were identified by 16 S rRNA gene sequencing (Table 1). Based on 16 S rRNA gene sequencing, WU-1 and WU-5 were identified as B. megaterium; WU-2 and WU-3 as B. velezensis; WU-4 and WU-9 as B. methylotrophicus; WU-6 as B. atrophaeus, WU-7 and WU-8 as B. aryabhattai; WU-10, WU-11 and WU-12 were B. amyloliquefaciens; and WU-13 as B. subtilis. Primary characterization of 13 isolates was also performed by different microbiological and biochemical tests (Table 1).

3.2.2. In vitro characterization of biocontrol factors of bacterial isolates In biocontrol properties, all bacterial isolates were tested for the production of siderophores, HCN and extra cellular enzymes (Table 2). The results showed that eight bacterial isolates formed halos in CASagar medium, indicating their abilities to produce siderophores. However, only two isolates (WU-2 and WU-8 strains) showed positive to HCN production test. In the extra cellular enzymes production test, no strain performed all eight extra cellular enzyme activities whereas eight isolates were able to produce six extra cellular enzymes (i.e. chitinase, glucanase, protease, cellulase, amylase, and lipase). Even all 13 strains produced cellulose, amylase and lipase, while a large majority of bacterial strains produced protease (i.e. 10 isolates or 76.9%) and glucanase (i.e. 9 isolates or 69.2%). Moreover, only two strains, WU-6 and WU-13, performed chitinase activity.

3.2. Characterization of selected bacteria 3.2.1. Direct PGP properties in selected bacterial isolates The thirteen bacteria were assayed for IAA production, nitrogen fixation (Table 2) and screened for phosphate solubilization activity

Table 1 Identification of the rhizobacterial isolates associated with pepper based on the 16 s rRNA gene sequence and microbial characteristics. Isolate

WU-1 WU-2 WU-3 WU-4 WU-5 WU-6 WU-7 WU-8 WU-9 WU-10 WU-11 WU-12 WU-13 a b c d

16s rRNA identity

Morphological characteristicsa

Biochemical testa

Carbohydrate utilizationa

Identification

Closest strain and ident

G.Sb

Spore

Motility

NARc

NIRd

M.R

V-P

CAT

Sucrose

Maltose

Xylose

Mannitol

Β-CD

B. B. B. B. B. B. B. B. B. B. B. B. B.

HQ840732.1 CP021888.1 CP021890.1 HQ844510.1 EF612719.1 EU194333.1 KX230137.1 HQ242766.1 HQ844484.1 JN086146.1 KF484682.1 AB301006.1 KX467568.1

+ + + + + + + + + + − − −

+ + + + − + − + − + − + +

− + − − + − + + − + + + +

− + + − − + + − + + + + +

+ − − + + − − + − − − − −

+ + − + + + − + + − − − −

− + + − − + + + + + + + −

+ + + + + + + + + + + + +

− + + + − + + + + + + + +

+ + + + + + + + + + + + +

+ + + − − − + − − + + + +

+ + + + + + + + + + + + +

+ + + + + − + + + + + − +

megaterium velezensis velezensis methylotrophicus megaterium atrophaeus aryabhattai aryabhattai methylotrophicus amyloliquefaciens amyloliquefaciens amyloliquefaciens subtilis

99% 100% 100% 99% 99% 99% 99% 99% 99% 99% 99% 99% 99%

+ represents production on plate or liquid assay; − no activity detected. G.S: Gram staining. NAR: Nitrate reduction. NIR: Nitrite reduction. 523

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Table 2 Overview of both direct and indirect plant growth promoting properties of the 13 bacterial isolates associated with pepper. Isolate

Direct PGP propertiesa,f PO43-b

WU-1 WU-2 WU-3 WU-4 WU-5 WU-6 WU-7 WU-8 WU-9 WU-10 WU-11 WU-12 WU-13 a b c d e f

− ++ − + ++ ++ − − + − ++ + −

N2 fixingc

− − − − − 0.86 ± 0.01 − − − − 0.79 ± 0.13 − −

Siderophore productiona

HCNa

IAAd

− − − 4.14 ± 0.05 − − − − − − − − −

Extra cellular enzyme productiona Chitinase

− + + − − + − + + + + + +

− + − − − − − + − − − − −

− − − − − + − − − − − − +

Protease

+ + + + − − − + + + + + +

Glucanase

+ − − − + + + + + + + + −

Cellulase

+ + + + + + + + + + + + +

Amylase

+ + + + + + + + + + + + +

Lipasee T20

T40

T80

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

+ + + + + + + + + + + + +

+ represents production on plate assay; −: no activity detected. PO43−: phosphate solubilization; −, no activity; +, 0.6–1.0 cm; ++, 1.0–2.0 cm. N2 fixing: nitrogen fixation (μg/mL). IAA: indole-3-acetic acid production (μg/mL). T20: Tween-20; T40: Tween-40; T80: Tween-80. values include standard error.

Fig. 1. Evaluation of primary plant growth promoting properties of thirteen strains. ACC deaminase activity (A), phosphate solubilizing ability (B), and Ammonia production (C) of different isolates in LB, NBRIP, and ADF liquid medium respectively. Bars represent means standard error. Different letters indicate average of three independent replicates with significant differences among different strains at the level P < 0.05 according to LSD Fisher's least significant difference test.

inoculated with the selected bacterial strains. The inoculation of WU-13 caused significant (P < 0.05) increase in fresh weight by 52.33%, dry weight by 80.66%, shoot height by 30.49% and root length by 143.12%, compared with those of non-inoculated control seedlings under salinity stress. In addition, WU-5 promoted the growth of

3.3. Bacterization of isolates in vitro-grown seedlings 3.3.1. Growth-promoting assays To evaluate the growth promoting ability of the 13 isolates on Honglong 23 under non-saline or saline stress, seedlings were 524

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Fig. 2. Evaluation of different plant growth promoting parameters to show the effect of thirteen strains inoculation on seedlings under non-saline and saline conditions (- and +, respectively). (A) dry weight, (B) fresh weight, (C) shoot and root length, and (D) chlorophyll of in vitro-grown plant leaves. CK represents noninoculated control. Bars represent means standard error. Different letters indicate average of twelve independent replicates with significant differences among different strains and saline treated peppers at the level P < 0.05 according to LSD Fisher's least significant difference test.

control under saline and non-saline conditions.

seedlings and led to statistically significant (P < 0.05) increase in fresh weight by 75.60% and dry weight by 86.68% under salinity stress, compared with those of non-inoculated seedlings. The results also showed that dry weight (Fig. 2A), fresh weight (Fig. 2B) and shoot and root length (Fig. 2C) of seedlings inoculated with WU-5 were not inhibited by salt stress. The results indicated that the inoculation of WU-5 may eliminate the observable detrimental effect of salt stress and have the same growth promotion effects as those under non-saline conditions. Thus, the results have indicated that those two isolates (WU-5 and WU-13) promoted biomass yield under saline conditions, compared with those non-inoculated control groups (Fig. 2).

3.3.4. Further growth-promoting assays under different salt stress As the salt concentration increased, the dry weight and fresh weight of non-inoculated peppers decreased (P < 0.05) continuously, but the fresh and dry weight, shoot length and root length of pepper inoculated all three strains were significantly higher than that of non-inoculated control under different saline stress (Fig. 4). Among of them, WU-9 showed better ability to promote plant growth under lower salt stress conditions (0 and 100 mM NaCl), followed by WU-5. Moreover, WU-5 inoculated seedlings registered significantly higher (P < 0.05) fresh weight, dry weight and shoot length in comparison to non-inoculated control and other two strains inoculated seedlings under salinity (200 and 300 mM NaCl) stress (Fig. 4 A-C).

3.3.2. Chlorophyll and soluble protein contents of seedlings Only the strain UW-13 significantly (P < 0.05) stimulated chlorophyll value of seedlings (increased by 44.50% over the non-inoculated control) under salt stress (Fig. 2D). Ten isolates significantly (P < 0.05) increased soluble protein content than the non-inoculated control without salt stress (Fig. 3C).

4. Discussion PGPR can promote plant growth by regulating nutritional and hormonal balance, producing plant growth regulators, and solubilizing nutrients for easy uptake by plants (Patten and Glick, 2002). As a result, plant growth may be enhanced by the application of microbial inoculation mainly depending on biological processes rather than on agrochemicals under stress conditions (Nadeem et al., 2014). To search for one or more PGPR candidates expected to promote plant growth, this study focused on the PGP properties of screened strains and their impacts on pepper growth and development under salt stress conditions. The results obtained corroborated with the fact that PGPR enhanced plant growth and development under salt stress. Thirteen bacterial strains with ACC deaminase activity were isolated, identified and

3.3.3. Antioxidant enzyme activities and proline content of seedlings POD activity was only significantly stimulated (P < 0.05) by WU-9 (Fig. 3A) under both non-saline and saline stress (Fig. 3B). The PPO activity was raised (P < 0.05) by WU-5, WU-9 and WU-13 inoculation without salinity stress. Seedlings almost accumulated higher amounts of proline under salt stress than non-saline condition (Fig. 3D). In addition, the different isolate inoculation treatments induced a generally significant increase (P < 0.05) of proline content of seedlings under salinity stress in the entire study (Fig. 3D). Among them, six strains (include WU-9) induced (P < 0.05) more proline than non-inoculated 525

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Fig. 3. Antioxidant levels assessed in leaves of seedlings bacterized with selected strains under non-saline and saline conditions (− and +, respectively). (A) POD, (B) PPO, (C) content of soluble protein, and (D) content of proline of in vitro-grown pepper. CK represents non-inoculated control. Bars represent means standard error. Different letters indicate average of twelve independent replicates with significant differences among different strains and saline treated peppers at the level P < 0.05 according to LSD Fisher's least significant difference test.

length by 146.52% in pot treatment amended with NaCl (Fig. 1). In addition, WU-9 also promoted root elongation by 39.34% and soluble-P concentration by 922.41 μg/mL in concordance with Glick (2012) and Pereira and Castro (2014). These results clearly indicated that the significant increase in root biomass of seedlings after inoculation with strains is closely related to better absorption of phosphate from potting substrate. The thirteen bacterial strains showed more than enough ACC deaminase activity to ameliorate saline stress because strains which have an enzyme activity more than 20 nmol of α-KB/mg/h could influence plant growth under salt stress (Glick, 2005; Penrose and Glick, 2003). The ACC deaminase activities, 172.50 nmol α-KB/mg/h in CDP-13 and 980 μmol α-KB/mg/h in IG-4, have been reported in Gerós et al. (2016) and Gontia-Mishra et al. (2016a), respectively. The highest deaminase activity was recorded in WU-9 as 47.32 μmol α-KB/mg/h. Alleviation of saline stress relies mainly on the decrease of stress-induced ethylene produced in plants under stress conditions (Glick, 2005). Furthermore, α-KB and ammonia, the breakdown products of ACC, serve as sources of nitrogen and energy respectively for the associated bacteria (Glick et al., 2007). Plant biomass was significantly higher with the inoculation of NH3producing isolates than non-producing isolates (Ghyselinck et al., 2013). Surprisingly, all isolates tested in this study were also able to produce large amounts of ammonia (Fig. 1); however, the apparent growth promoting effect of ammonia was not found. Two strains from selected isolates exhibited HCN production and nine manifested siderophore production. The antagonistic activity against Phytophthora infestans of HCN-producing isolates was

systematically investigated for their PGP properties and effects on seedling growth and development under saline stress. The tested isolates were identified by using 16S rRNA gene sequencing (Table 1). The bacterial strains showed diversities at the species-to-strain level. Nevertheless, diversity at the genus level was all restricted to Bacillus. With respect to PGP properties, bacterial strains were confirmed to have a series of relevant properties, including nitrogen fixation; IAA, siderophore, and NH3 production; inorganic phosphate solubilization; ACC deaminase and extra cellular enzyme activities (Table 2 and Fig. 1). IAA production is a prominent feature of PGP activity because IAA modulates plant root growth and development (Gontia-Mishra et al., 2016a). In the present study, only WU-4 strain showed an activity for IAA production. Analogously, rhizobacteria with nitrogen fixing capacity reduced toxicity under environment stress and improved plant growth, suggesting their potential role for biological fertilizers (Nadeem et al., 2014). Nitrogen fixation capacity was demonstrated only by two isolates (WU-6 and WU-11). However, there was no significant increase in biomass of seedlings inoculated with WU-6 and WU11. Phosphorus deficiency is widespread in soil, and phosphorus fertilizers are widely applied to maintain crop production (Pereira and Castro, 2014). However, plants can only utilize a small part of phosphatic fertilizers applied into the agricultural soils, and the rest is converted into phosphorus-containing compounds of low solubility (Costa et al., 2012; Glick, 2012). The best phosphate-solubilizing bacteria WU-5, which soluble phosphorus ability reached to 785.63 μg/mL in liquid inorganic phosphorus medium, increased (P < 0.05) root 526

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Fig. 4. Further growth-promoting ability assays of three selected strains under different saline conditions. (A) fresh weight, (B) dry weight, (C) shoot length, and (D) root length of in vitro-grown pepper. Bars represent means standard error. Different letters indicate average of fifteen independent replicates with significant differences among different strains and saline treated peppers at the level P < 0.05 according to LSD Fisher's least significant difference test.

faba (Qados, 2011). This finding indicated that WU-13 had the ability to induce chlorophyll accumulation under salt stress. Moreover, salt stress enhanced the rhizosphere osmotic pressure, thereby reducing the water flow toward roots (Gontia-Mishra et al., 2016b). The soluble protein content was significantly stimulated (P < 0.05) in non-inoculated plants by salt stress. It was speculated that plants increased their osmotic potential by accumulating compatible osmolytes, such as proline and total soluble sugar, to maintain root water uptake and reduce the negative impact of salt stress (Porcel and Ruiz-Lozano, 2004). The increase in soil salinity causes membrane degradation, lipid peroxidation and several other physiological and biochemical alteration in plants. These events induce the cellular generation of reactive oxygen species (ROS), such as hydroxyl radicals (•OH), superoxide (O2-), and hydrogen peroxide (H2O2), which eventually causes cell death (Gerós et al., 2016). Antioxidant enzymes were differentially activated in seedlings depending on the bacteria. The differences in antioxidant enzyme activities in seedlings among microbial treatments may also be related to hormonal changes brought about by the microbial treatments (Pinter et al., 2017). Supplementation of NaCl tended to increase the activity of PPO and POD in non-inoculated control, whereas the enzymatic activity was only increased (P < 0.05) by WU-9 or even reduced the POD activity by six strains under salt stress. These results indicate that the relief of salt stress was not due to an increase in the activity of antioxidant enzymes. Proline has been established as a multi-functional molecule, which accumulates in high concentrations in response to a variety of abiotic stresses (Kavi Kishor and Sreenivasulu, 2014). Considered not only as a compatible solute and osmoprotectant but also a hydroxyl radical scavenger, proline has been reported to reduce the salt stress-induced enzyme denaturation by scavenging reactive oxygen species (Gerós et al., 2016). Proline homeostasis is important to maintain growth under long-term stress (Kavi Kishor and Sreenivasulu, 2014). During a

significantly higher than that of non-producing strains (Ghyselinck et al., 2013). Siderophore producing bacterial strains not only inhibited fungal growth through competition for iron but also induced systemic resistance in plants and promoted plant growth indirectly (Ahmad et al., 2008; Ghyselinck et al., 2013). Previous studies have shown that the antifungal activity of PGPR indicated a close relationship with siderophore production, HCN production, or synergistic interaction of the two or with other metabolites (Ahmad et al., 2008). The tested isolates also exhibited antagonistic properties against plant pathogens due to their abilities of synthesize extracellular enzymes, such as chitinase and glucanase (Kumar et al., 2017). The application of PGPR with the ability to produce cell-wall degrading enzymes as bio-inoculants in soil-borne diseases infested soils will confer a great advantage for promoting plant growth (Ghyselinck et al., 2013). The rhizobacteria with extracellular enzyme synthesis could have better ability to withstand salinity as compared with other PGPR (Kumar et al., 2017). In addition, two strains (WU-5 and WU-13) stimulated a significant increase (P < 0.05) in fresh weight, dry weight and root length of seedlings compared with the non-inoculated controls under saline conditions (Fig. 2). Obviously, the results suggested that the two isolates can be of great value in better promoting seedlings growth with a higher biomass production compared with non-inoculated controls under salt stress. These observations also indicated that the two bacterial strains (WU-5 and WU-13) selected could be interesting targets for the development of new commercial microbial agents. Moreover, we supposed that plant growth promotion might be due to the production of phosphate solubilizing ability, such as WU-5, or synergistic interaction of these factors or with other metabolites. Inoculation of strain WU-13 significantly stimulated (P < 0.05) the accumulation of chlorophyll in seedlings under saline stress. However, the chlorophyll content decreased in plants grown in previous reports under salinity condition, such as paddy (Kumar et al., 2017) and Vicia

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stress episode, proline is accumulated, which can be degraded to provide a supply of energy to drive plant growth once the stress is relieved (Calvo-Polanco et al., 2016; Kavi Kishor and Sreenivasulu, 2014). Saline stress might have resulted in increased proline content due to increased generation of ROS and rhizosphere osmotic pressure. Moreover, proline content was significantly higher (P < 0.05) in seedlings inoculated with 11 isolates as compared with non-inoculated control plants under salt stress (Fig. 3D). The accumulation of proline also alleviates the reduction of antioxidant enzyme activities caused by saline stress (Penrose and Glick, 2003). In addition, the differences of proline accumulation among microbial treatments may also be associated with hormonal changes in pepper. Salicylic acid, auxins and abscisic acid (ABA) are known to up-regulate proline synthesis, whereas cytokinin down-regulates proline accumulation (Calvo-Polanco et al., 2016; Kavi Kishor and Sreenivasulu, 2014). All together, these results indicated that the inoculation with the selected bacterial strains produced alleviation in saline stressed pepper, due to an increase in proline content. Moreover, the results suggested that WU-9, showing high level of proline production and antioxidant enzyme activities, can maintain the growth and development of seedlings better than non-inoculated controls under salt stress. Based on the analysis of plant growth promoting activity of 13 strains under salt stress, three isolates (WU-5, WU-9 and WU-13) were selected to further growth promoting ability study under different saline conditions. According to the variation of fresh weight, dry weight, shoot length and root length of peppers under different salt stress, all three selected strains can promote (P < 0.05) plant growth and alleviate (P < 0.05) salt stress to varying degrees (Fig. 4). These isolates can be of great value in maintaining the growth and development of seedlings on saline lands.

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